A simple and efficient agroinfiltration method in coffee leaves (Coffea arabica L.): assessment of factors affecting transgene expression

Abstract

The establishment of a simple, rapid and efficient transient expression system is a necessary tool for the functional validation of candidate genes in coffee biotechnology. The effects of Agrobacterium strain, age of the donor plant, infiltration method, and infiltration medium on transgene expression in detached coffee leaves were evaluated. Regarding the effect of Agrobacterium strain, the expression of uidA was higher in GV3101-treated coffee disks than in LBA4404 and ATHV-treated samples. On the other hand, transient expression of uidA was significantly higher in leaf disks from young plants (6-weeks-old) (13.1 ± 1.4%) than in mature tissue (12-weeks-old) (1.6 ± 1.2%). Transient uidA expression was higher in detached coffee leaf disks from young plants infiltrated with one injection of 15 µL of Agrobacterium strain GV3101::1303 suspended in MS salts supplemented with 30 g/L sucrose, 1.9 g/L MES and 200 uM AS with subsequent sanding of the abaxial epidermis. Using the optimized protocol, expression of the uidA gene was observed 6, 24 and 48 h and 5 weeks after bacterial injection. DNA was extracted from coffee disks with positive GUS expression and specific mgfp5 and uidA fragments were amplified 5 weeks post-agroinfiltration. On the other hand, using the optimized protocol, a specific cry10Aa (500 bp) fragment was amplified in the agro-infiltrated coffee leaf disks 5 weeks post-agroinfiltration with the plasmid pB427-35S-cry10Aa. Moreover, the expression of the gene cry10Aa in two infiltrated coffee leaf disks was verified by RT-PCR and an expected 500 bp fragment was amplified.

Transient Gene Expression is an Effective Experimental Tool for the Research into the Fine Mechanisms of Plant Gene Function: Advantages, Limitations, and Solutions

“…This suggests that AGL1 is more aggressively disposed for infection or, presumably, has a more efficient bacteria-encoded mechanism for T-DNA transfer as compared with the other two tested strains [ 50 ]. Similar studies have been performed for several other plant species [ 4 , 80 ].…”

“…As has been convincingly demonstrated, the performance of younger leaves in the transient expression experiments is better; however, since they are, as a rule, of very small size, the first–fourth leaves from the top are usually selected. In addition, the leaves of a flowering plant are not recommended for transient expression experiments because of a lowered transcription and translation levels in the host cells in older leaves and during blossoming, which may have negative effects on the expression efficiency of target genes [ 79 , 80 , 81 ].…”

A high‑efficiency Agrobacterium tumefaciens‑mediated transient expression system in the leaves of Spinacia oleracea

“…), tobacco and tomato and A. annua [26,32]. Besides, the development stage of the plant also had an impact on the transient expression levels [45]. We also con rmed that G1 showed the highest uorescence intensity, and G1 at the four-leaf stage was more suitable than that at the six-leaf stage (Figs.…”

CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane

“…Methods for efficient transformation of both C. arabica and C. canephora species mostly include particle bombardment ( Ribas et al., 2005 ; Albuquerque et al., 2009 ; De Guglielmo-Cróquer et al., 2010 ), Agrobacterium -mediated transformation of embryogenic callus ( Ribas et al., 2011 ), hairy roots ( Alpizar et al., 2006 ), agroinfiltration of mature leaves ( Vargas-Guevara et al., 2018 ) and protoplast electroporation ( Fernandez and Menéndez-Yuffá, 2003 ) ( Figure 4 ). Overall transformation efficiency depends on several parameters, reviewed by Mishra and Slater (2012) and Etienne et al.…”